The purpose of this project is to determine whether changing the location of several glycosyl transferases within the Golgi alters the amount and types of oligosaccharides they make. SLeX and LeX occur on polylactosamine repeats of O-linked sugar chains. Core 2 GicNActransferase is required to initiate their synthesis, and this enzyme is thought to reside in the "mid- Golgi". An alpha2,6 sialyl transferase that makes sialyl Tn antigen by adding Sia to the Ga1NAc initiating O-linked chains is thought to occur in an "earlier" Golgi compartment. Addition of this residue blocks Core 2 GlcNAc transferase. A second alpha2,6sialyl transferase terminates extended polylactosamine chains and should be located in the "late Golgi". Since the putative targeting domains of all Golgi enzymes are thought to lie in the cytoplasmic, transmembrane and stem regions, the catalytic domain can be shuffled to different locations by using different targeting domains. The effects on glycosylation will be monitored by measuring the amount of polylactosamine and sialylated produced made in the different constructs. However, traditional methods to localize the transferases (e.g., immunolocalization) give a low resolution picture of the Golgi and cannot define functionally active glycosyl transferases. We propose to use two related biochemical methods in mapping functionally active glycosyl transferases as they move around the Golgi. Both methods rely on the ability of Golgi to transport and concentrate radiolabeled sugar nucleotides to glycosylate exogenous added synthetic and/or endogenous acceptors. First, synthetic glycoside acceptors (e.g., GaINAcalpha-MU) that prime complex glycan synthesis in living cells will be added to Golgi preparations along with limited amounts of radiolabeled sugar nucleotides (e.g., UDP-[3H]Gal). The acceptors enter all compartments, but are labeled only in those compartments that have glycosyl transferases. and the transport the donors (e.g. O-linked core specific betaGal transferase I, forming GalbetaGalNAcalphaMU). Additional glycosylations (e.g., Core 2 GlcNAc residues) can occur on the same labeled acceptor only if the next transferase is to-localized in that same functional compartment. Second, the same transported donors can also label endogenous products. However, within the compartment where the glycosides were made the amount of transported sugar nucleotide is now limited; there is less to glycosylate the trace amounts of endogenous products awaiting 3H-Gal addition (e.g., N- linked oligosaccharides). As the amount of glycoside product increases, glycosylation of the endogenous acceptors decreases in proportional to the degree of enzyme co-localization. Detailed structural analysis of products made on both added acceptors and endogenous acceptors indicates which transferases are co-localized with each other. This method sketches a detailed map of neighboring functional transferases of several glycosylation pathways within a compartment, and it should reveal change in transferase localization. Results obtained by these methods will be compared to those obtained by traditional immunological techniques to yield an enhanced picture of enzyme localization.